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1.
PLoS Biol ; 17(8): e3000203, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31430272

RESUMO

Zebrafish dorsal forerunner cells (DFCs) undergo vigorous proliferation during epiboly and then exit the cell cycle to generate Kupffer's vesicle (KV), a ciliated organ necessary for establishing left-right (L-R) asymmetry. DFC proliferation defects are often accompanied by impaired cilia elongation in KV, but the functional and molecular interaction between cell-cycle progression and cilia formation remains unknown. Here, we show that chemokine receptor Cxcr4a is required for L-R laterality by controlling DFC proliferation and KV ciliogenesis. Functional analysis revealed that Cxcr4a accelerates G1/S transition in DFCs and stabilizes forkhead box j1a (Foxj1a), a master regulator of motile cilia, by stimulating Cyclin D1 expression through extracellular regulated MAP kinase (ERK) 1/2 signaling. Mechanistically, Cyclin D1-cyclin-dependent kinase (CDK) 4/6 drives G1/S transition during DFC proliferation and phosphorylates Foxj1a, thereby disrupting its association with proteasome 26S subunit, non-ATPase 4b (Psmd4b), a 19S regulatory subunit. This prevents the ubiquitin (Ub)-independent proteasomal degradation of Foxj1a. Our study uncovers a role for Cxcr4 signaling in L-R patterning and provides fundamental insights into the molecular linkage between cell-cycle progression and ciliogenesis.


Assuntos
Cílios/metabolismo , Fatores de Determinação Direita-Esquerda/metabolismo , Receptores CXCR4/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Padronização Corporal/genética , Ciclo Celular/fisiologia , Divisão Celular , Proliferação de Células , Quimiocinas/metabolismo , Embrião não Mamífero/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese , Receptores CXCR4/fisiologia , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
2.
Fish Shellfish Immunol ; 92: 224-229, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200068

RESUMO

Fibroblast growth factor receptor (FGFR) 3 is one of the four distinct membrane-spanning tyrosine kinases required for proper skeletal development. In fish, the role of FGFR3 is still unclear. In this article, we reveal that zebrafish FGFR3 is a negative regulator of interferon (IFN) production in the innate immune response by suppressing the activity of TANK-binding kinase 1 (TBK1) in the process of virus infection. qPCR experiments demonstrate that the transcriptional level of cellular FGFR3 was upregulated by infection with spring viremia of carp virus (SVCV), indicating that FGFR3 might be involved in the process of host cell response to viral infection. Then, overexpression of FGFR3 significantly impeded the IFN promoter activity induced by a stimulator. In addition, the capabilities of a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) system to activate IFN promoter were decreased during the overexpression of FGFR3. Subsequently, FGFR3 decreased the phosphorylation of interferon regulatory factor 3 (IRF3) and mediator of IRF3 activation (MITA) by TBK1. These findings suggest that zebrafish FGFR3 is a negative regulator of IFN by attenuating the kinase activity of TBK1, leading to the suppression of IFN expression.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata/genética , Interferons/genética , Proteínas Serina-Treonina Quinases/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Interferons/metabolismo , Proteínas Serina-Treonina Quinases/imunologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/imunologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Transdução de Sinais/imunologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/fisiologia
3.
Nat Commun ; 10(1): 2024, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048699

RESUMO

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.


Assuntos
Lesão Renal Aguda/patologia , Cálcio/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Lesão Renal Aguda/genética , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Túbulos Renais Proximais/citologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologia
4.
Nat Commun ; 10(1): 1551, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948728

RESUMO

The segregation of eukaryotic genomes into euchromatin and heterochromatin represents a fundamental and poorly understood process. Here, we demonstrate that genome-wide establishment of heterochromatin is triggered by the maternal to zygotic transition (MZT) during zebrafish embryogenesis. We find that prior to MZT, zebrafish lack hallmarks of heterochromatin including histone H3 lysine 9 trimethylation (H3K9me3) and condensed chromatin ultrastructure. Global establishment of heterochromatic features occurs following MZT and requires both activation of the zygotic genome and degradation of maternally deposited RNA. Mechanistically, we demonstrate that zygotic transcription of the micro RNA miR-430 promotes degradation of maternal RNA encoding the chromatin remodeling protein Smarca2, and that clearance of Smarca2 is required for global heterochromatin establishment in the early embryo. Our results identify MZT as a key developmental regulator of heterochromatin establishment during vertebrate embryogenesis and uncover functions for Smarca2 in protecting the embryonic genome against heterochromatinization.


Assuntos
Desenvolvimento Embrionário/genética , Heterocromatina/genética , Peixe-Zebra/embriologia , Animais , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Transcrição Genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia
5.
Psychopharmacology (Berl) ; 236(7): 2049-2058, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30820632

RESUMO

RATIONALE: The endocannabinoid system (ECS) comprises the cannabinoids anandamide and 2-arachidonoylglycerol and the cannabinoid receptors 1 and 2 (Cnr1 and Cnr2). The function of these receptors in relation to zebrafish larval behavior is poorly understood, even though the zebrafish larva has become a versatile animal model in biomedical research. OBJECTIVES: The objective of the present study is to characterize the function of Cnr1 and Cnr2 in relation to behavior in zebrafish. METHODS: Behavioral analysis of zebrafish larvae was performed using a visual motor response (VMR) test, which allows locomotor activity to be determined under basal conditions and upon a dark challenge. RESULTS: Treatment with the non-specific Cnr agonists WIN55,212-2 and CP55,940 resulted in a decrease in locomotion. This was observed for both basal and challenge-induced locomotion, although the potency for these two effects was different, which suggests different mechanisms of action. In addition, WIN55,212-2 increased the reaction time of the startle response after the dark challenge. Using the Cnr1 antagonist AM251 and a cnr1-/- mutant line, it was shown that the effects were mediated by Cnr1 and not Cnr2. Interestingly, administration of the antagonist AM251 alone does not have an effect on locomotion, which indicates that endogenous cannabinoid activity does not affect locomotor activity of zebrafish larvae. Upon repeated dark challenges, the WIN55,212-2 effect on the locomotor activity decreased, probably due to desensitization of Cnr1. CONCLUSIONS: Taken together, these results show that Cnr1 activation by exogenous endocannabinoids modulates both basal and challenge-induced locomotor activity in zebrafish larvae and that these behavioral effects can be used as a readout to monitor the Cnr1 responsiveness in the zebrafish larva model system.


Assuntos
Adaptação à Escuridão/fisiologia , Larva/metabolismo , Locomoção/fisiologia , Receptor CB1 de Canabinoide/fisiologia , Receptor CB2 de Canabinoide/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Ácidos Araquidônicos/farmacologia , Canabinoides/farmacologia , Adaptação à Escuridão/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endocanabinoides/farmacologia , Glicerídeos/farmacologia , Larva/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Pirazóis/farmacologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Peixe-Zebra , Proteínas de Peixe-Zebra/agonistas
6.
Development ; 146(6)2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30837221

RESUMO

Hematopoiesis maintains the entire blood system, and dysregulation of this process can lead to malignancies (leukemia), immunodeficiencies or red blood cell diseases (anemia, polycythemia vera). We took advantage of the zebrafish model that shares most of the genetic program involved in hematopoiesis with mammals to characterize a new gene of unknown function, si:ch73-299h12.2, which is expressed in the erythroid lineage during primitive, definitive and adult hematopoiesis. This gene, required during primitive and definitive erythropoiesis, encodes a C2H2 zinc-finger protein that inhibits BMP signaling. We therefore named this gene blood-inducing factor 1 and BMP inhibitory factor 1 (bif1). We identified a bif1 ortholog in Sinocyclocheilus rhinocerous, another fish, and in the mouse genome. Both genes also inhibit BMP signaling when overexpressed in zebrafish. In conclusion, we have deorphanized a new zebrafish gene of unknown function: bif1 codes for a zinc-finger protein that inhibits BMP signaling and also regulates primitive erythropoiesis and definitive hematopoiesis.


Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Núcleo Celular/metabolismo , Eritropoese/genética , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Dedos de Zinco
7.
PLoS One ; 14(3): e0211399, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30861003

RESUMO

The zebrafish has become a valuable model for examining ocular lens development, physiology and disease. The zebrafish cloche mutant, first described for its loss of hematopoiesis, also shows reduced eye and lens size, interruption in lens cell differentiation and a cataract likely caused by abnormal protein aggregation. To facilitate the use of the cloche mutant for studies on cataract development and prevention we characterized variation in the lens phenotype, quantified changes in gene expression by qRT-PCR and RNA-Seq and compared the ability of two promoters to drive expression of introduced proteins into the cloche lens. We found that the severity of cloche embryo lens cataract varied, while the decrease in lens diameter and retention of nuclei in differentiating lens fiber cells was constant. We found very low expression of both αB-crystallin genes (cryaba and cryabb) at 4 days post fertilization (dpf) by both qRT-PCR and RNA-Seq in cloche, cloche sibling and wildtype embryos and no significant difference in αA-crystallin (cryaa) expression. RNA-Seq analysis of 4 dpf embryos identified transcripts from 25,281 genes, with 1,329 showing statistically significantly different expression between cloche and wildtype samples. Downregulation of eight lens ß- and γM-crystallin genes and 22 retinal related genes may reflect a general reduction in eye development and growth. Six stress response genes were upregulated. We did not find misregulation of any known components of lens development gene regulatory networks. These results suggest that the cloche lens cataract is not caused by loss of αA-crystallin or changes to lens gene regulatory networks. Instead, we propose that the cataract results from general physiological stress related to loss of hematopoiesis. Our finding that the zebrafish αA-crystallin promoter drove strong GFP expression in the cloche lens demonstrates its use as a tool for examining the effects of introduced proteins on lens crystallin aggregation and cataract prevention.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Catarata/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Cristalinas/genética , Cristalinas/fisiologia , Modelos Animais de Doenças , Hematopoese/fisiologia , Cristalino/metabolismo , Cristalino/fisiologia , Mutação , Fenótipo , Peixe-Zebra/genética , Cadeia A de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/genética
8.
Biochim Biophys Acta Gene Regul Mech ; 1862(4): 472-485, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30840854

RESUMO

The multidomain RNase III endoribonuclease DICER is required for the generation of most functional microRNAs (miRNAs). Loss of Dicer affects developmental processes at different levels. Here, we characterized the zebrafish Dicer1 mutant, dicer1sa9205, which has a single point mutation induced by N-ethyl-N-nitrosourea mutagenesis. Heterozygous dicer1sa9205 developed normally, being phenotypically indistinguishable from wild-type siblings. Homozygous dicer1sa9205 mutants display smaller eyes, abnormal craniofacial development and aberrant pigmentation. Reduced numbers of both iridophores and melanocytes were observed in the head and ventral trunk of dicer1sa9205 homozygotes; the effect on melanocytes was stronger and detectable earlier in development. The expression of microphthalmia-associated transcription factor a (mitfa), the master gene for melanocytes differentiation, was enhanced in dicer1-depleted fish. Similarly, the expression of SRY-box containing gene 10 (sox10), required for mitfa activation, was higher in mutants than in wild types. In silico and in vivo analyses of either sox10 or mitfa 3'UTRs revealed conserved potential miRNA binding sites likely involved in the post-transcriptional regulation of both genes. Based on these findings, we propose that dicer1 participates in the gene regulatory network governing zebrafish melanocyte differentiation by controlling the expression of mitfa and sox10.


Assuntos
Cartilagem/anormalidades , Melanócitos/citologia , Ribonuclease III/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Regiões 3' não Traduzidas , Animais , Apoptose , Cartilagem/crescimento & desenvolvimento , Embrião não Mamífero/anormalidades , Embrião não Mamífero/anatomia & histologia , Regulação da Expressão Gênica , Cabeça , Larva/anatomia & histologia , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Mutação , Crista Neural/citologia , Ribonuclease III/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Dis Model Mech ; 12(3)2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30765415

RESUMO

Microglia are brain-resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive the migration of yolk sac macrophages (YSMs) into the embryonic brain, or regulate their acquisition of specialized properties, are currently known. Here, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new microglia regulators using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency and conserved microglia features. We targeted putative microglia regulators, by Cas9/gRNA complex injections, followed by Neutral-Red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of zebrafish colony-stimulating factor 1 receptor (Csf1r) ligand, Il34, caused reduced microglia numbers. Previous studies on the role of IL34 in microglia development in vivo were ambiguous. Our data, and a concurrent paper, show that, in zebrafish, il34 is required during the earliest seeding of the brain by microglia. Our data also indicate that Il34 is required for YSM distribution to other organs. Disruption of the other Csf1r ligand, Csf1, did not reduce microglia numbers in mutants, whereas overexpression increased the number of microglia. This shows that Csf1 can influence microglia numbers, but might not be essential for the early seeding of the brain. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of YSMs to target organs, validating our reverse genetic screening pipeline in zebrafish.This article has an associated First Person interview with the joint first authors of the paper.


Assuntos
Encéfalo/metabolismo , Testes Genéticos , Interleucinas/metabolismo , Macrófagos/metabolismo , Genética Reversa , Saco Vitelino/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Encéfalo/crescimento & desenvolvimento , Sistemas CRISPR-Cas/genética , Contagem de Células , Proliferação de Células , Interleucinas/genética , Interleucinas/fisiologia , Microglia/metabolismo , Mutação/genética , Proteínas de Peixe-Zebra/genética
10.
PLoS Genet ; 15(2): e1007982, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30779743

RESUMO

Following injury, axons of the peripheral nervous system have retained the capacity for regeneration. While it is well established that injury signals require molecular motors for their transport from the injury site to the nucleus, whether kinesin and dynein motors play additional roles in peripheral nerve regeneration is not well understood. Here we use genetic mutants of motor proteins in a zebrafish peripheral nerve regeneration model to visualize and define in vivo roles for kinesin and dynein. We find that both kinesin-1 and dynein are required for zebrafish peripheral nerve regeneration. While loss of kinesin-1 reduced the overall robustness of axonal regrowth, loss of dynein dramatically impaired axonal regeneration and also reduced injury-induced Schwann cell remodeling. Chimeras between wild type and dynein mutant embryos demonstrate that dynein function in neurons is sufficient to promote axonal regrowth. Finally, by simultaneously monitoring actin and microtubule dynamics in regenerating axons we find that dynein appears dispensable to initiate axonal regrowth, but is critical to stabilize microtubules, thereby sustaining axonal regeneration. These results reveal two previously unappreciated roles for dynein during peripheral nerve regeneration, initiating injury induced Schwann cell remodeling and stabilizing axonal microtubules to sustain axonal regrowth.


Assuntos
Dineínas/fisiologia , Regeneração Nervosa/fisiologia , Nervos Periféricos/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Axônios/ultraestrutura , Dineínas/genética , Cinesina/genética , Cinesina/fisiologia , Mutação , Regeneração Nervosa/genética , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/patologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Células de Schwann/citologia , Células de Schwann/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética
11.
Dev Dyn ; 248(4): 284-295, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30801852

RESUMO

BACKGROUND: During heart morphogenesis, the cardiac chambers undergo ballooning: a process involving regionalized elongation of cardiomyocytes. Cardiomyocyte shape changes require reorganization of the actin cytoskeleton; however, the genetic regulation of this process is not well understood. RESULTS: From a forward genetic screen, we identified the zebrafish uq 23ks mutant which manifests chamber ballooning defects. Whole-genome sequencing-mapping identified a truncating mutation in the gene, myo5b. myo5b encodes an atypical myosin required for endosome recycling and, consistent with this, increased vesicles were observed in myo5b mutant cardiomyocytes. Expression of RFP-Rab11a (a recycling endosome marker) confirmed increased recycling endosomes in cardiomyocytes of myo5b mutants. To investigate potential cargo of MyoVb-associated vesicles, we examined the adherens junction protein, N-cadherin. N-cadherin appeared mispatterned at cell junctions, and an increase in the number of intracellular particles was also apparent. Co-localization with RFP-Rab11a confirmed increased N-cadherin-positive recycling endosomes, demonstrating N-cadherin trafficking is perturbed in myo5b mutants. Finally, phalloidin staining showed disorganized F-actin in myo5b cardiomyocytes, suggesting the cytoskeleton fails to remodel, obstructing chamber ballooning. CONCLUSIONS: MyoVb is required for cardiomyocyte endosomal recycling and appropriate N-cadherin localization during the onset of chamber ballooning. Cardiomyocytes lacking MyoVb are unable to reorganize their actin cytoskeleton, resulting in failed chamber ballooning. Developmental Dynamics 248:284-295, 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Caderinas/metabolismo , Citoesqueleto/ultraestrutura , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Miosina Tipo V/fisiologia , Animais , Forma Celular , Citoesqueleto/metabolismo , Endossomos/metabolismo , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Miosina Tipo V/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
12.
Mol Biol Cell ; 30(3): 293-301, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30516436

RESUMO

The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet to be identified. We have determined that Fer1l6, a member of the ferlin family of genes, is a novel factor in zebrafish development. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein-based assays indicate that Fer1l6 colocalizes with syntaxin 4 and vinculin, and that the putative C2 domains interact with lipid membranes. We conclude that Fer1l6 has diverged from other vertebrate ferlins to play an essential role in zebrafish skeletal and cardiac muscle development.


Assuntos
Desenvolvimento Muscular , Músculos/embriologia , Músculos/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Camundongos , Morfolinos/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Desenvolvimento Muscular/genética , Músculos/ultraestrutura , Mutação/genética , Proteínas Qa-SNARE/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteínas de Transporte Vesicular/genética , Vinculina/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
13.
Dev Dyn ; 248(2): 173-188, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30444277

RESUMO

BACKGROUND: Many human gene mutations have been linked to congenital heart disease (CHD), yet CHD remains a major health issue worldwide due in part to an incomplete understanding of the molecular basis for cardiac malformation. RESULTS: Here we identify the orthologous mouse Pou6f1 and zebrafish pouC as POU homeodomain transcription factors enriched in the developing heart. We find that pouC is a multi-functional transcriptional regulator containing separable activation, repression, protein-protein interaction, and DNA binding domains. Using zebrafish heart development as a model system, we demonstrate that pouC knockdown impairs cardiac morphogenesis and affects cardiovascular function. We also find that levels of pouC expression must be fine-tuned to enable proper heart formation. At the cellular level, we demonstrate that pouC knockdown disrupts atrioventricular canal (AVC) cardiomyocyte maintenance, although chamber myocyte specification remains intact. Mechanistically, we show that pouC binds a bmp4 intronic regulatory element to mediate transcriptional activation. CONCLUSIONS: Taken together, our study establishes pouC as a novel transcriptional input into the regulatory hierarchy that drives AVC morphogenesis in zebrafish. We anticipate that these findings will inform future efforts to explore functional conservation in mammals and potential association with atrioventricular septal defects in humans. Developmental Dynamics 248:173-188, 2019. © 2018 Wiley Periodicals, Inc.


Assuntos
Proteína Morfogenética Óssea 4/genética , Regulação da Expressão Gênica no Desenvolvimento , Septos Cardíacos/crescimento & desenvolvimento , Fatores do Domínio POU/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Coração/embriologia , Coração/crescimento & desenvolvimento , Defeitos dos Septos Cardíacos , Septos Cardíacos/embriologia , Camundongos , Fatores do Domínio POU/metabolismo , Ligação Proteica , Fatores de Transcrição , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
14.
Neurotoxicology ; 71: 31-38, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30521821

RESUMO

Gonadotropin releasing hormone (GnRH) neurons in the brain are the main controllers of reproduction and reproductive behavior in most vertebrates, and are susceptible to endocrine disruption by different bisphenols. While the endocrine disrupting properties of bisphenol A have been well documented, commonly used analogues such as bisphenol F (BPF) are not as well studied. In this study we examined the effects of early, low-dose, chronic BPF exposure on the development of the GnRH neural system in the zebrafish embryo. Using a transgenic zebrafish model system with GnRH3 neurons tagged with green fluorescent protein (GFP), developing GnRH neurons in both the terminal nerve (TN) and preoptic area (POA) were observed. These are neuronal populations with the former associated with allied reproductive behaviors and the latter associated with pituitary-gonadal axis control. Embryos were exposed in vitro to 0.25, 0.5 and 1 µM BPF from fertilization to 3 days post fertilization (dpf). At 0.25 µM BPF exposure, both POA- and TN- GnRH3 neurons showed significant reductions in neural area at 2 dpf that did not persist to 3 dpf. The higher BPF doses did not show neuron size differences at 2 dpf, but showed reduction in TN-GnRH3 neuron area at 3 dpf. These effects of BPF were closely mimicked by different doses of estradiol. An estrogen antagonist, ICI, mitigated BPF effects on the embryo. This is the first study to show that BPF affects the developing GnRH neural system via an estrogen-mediated pathway.


Assuntos
Compostos Benzidrílicos/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Estradiol/fisiologia , Hormônio Liberador de Gonadotropina/fisiologia , Neurônios/efeitos dos fármacos , Fenóis/toxicidade , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/efeitos dos fármacos , Estradiol/administração & dosagem , Feminino , Masculino , Neurônios/patologia , Neurônios/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologia
15.
Invest Ophthalmol Vis Sci ; 59(15): 6089-6101, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30592497

RESUMO

Purpose: Replacing cone photoreceptors, the units of the retina necessary for daytime vision, depends upon the successful production of a full variety of new cones from, for example, stem cells. Using genetic experiments in a model organism with high cone diversity, zebrafish, we map the intersecting effects of cone development factors gdf6a, tbx2b, and thrß. Methods: We investigated these genes of interest by using genetic combinations of mutants, gene knockdown, and dominant negative gene expression, and then quantified cone subtype outcomes (which normally develop in tightly regulated ratios). Results: Gdf6a mutants have reduced blue cones and, discovered here, reduced red cones. In combined gdf6a/tbx2b disruption, the loss of gdf6a in heterozygous tbx2b mutants reduced UV cones. Intriguingly, when we disrupted thrß in gdf6a mutants by using a thrß morpholino, their combined early disruption revealed a lamination phenotype. Disrupting thrß activity via expression of a dominant negative thrß (dnthrß) at either early or late retinal development had differential outcomes on red cones (reduced abundance), versus UV and blue cones (increased abundance). By using dnthrß in gdf6a mutants, we revealed that disrupting thrß activity did not change gdf6a mutant cone phenotypes. Conclusions: Gdf6a loss directly affects blue and red cones and indirectly affects UV cones by increasing sensitivity to additional disruption, such as reduced tbx2b, resulting in fewer UV cones. The effects of thrß change through photoreceptor development, first promoting red cones and restricting UV cones, and later restricting UV and blue cones. The effects of gdf6a on UV, blue, and red cone development overlap with, but likely supersede, those of thrß.


Assuntos
Diferenciação Celular/fisiologia , Fator 6 de Diferenciação de Crescimento/fisiologia , Retina/embriologia , Células Fotorreceptoras Retinianas Cones/citologia , Células-Tronco/fisiologia , Proteínas com Domínio T/fisiologia , Receptores beta dos Hormônios Tireóideos/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Regulação da Expressão Gênica/fisiologia , Inativação Gênica , Técnicas de Genotipagem , Imuno-Histoquímica , Modelos Animais , Células Fotorreceptoras de Vertebrados/fisiologia , Polimorfismo de Fragmento de Restrição , Peixe-Zebra
16.
Invest Ophthalmol Vis Sci ; 59(15): 6057-6066, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30577041

RESUMO

Purpose: Retinal degenerative diseases can progress to severe reductions of vision. In general, the changes are permanent in higher vertebrates, including humans; however, retinal regeneration can occur in lower vertebrates, such as amphibians and teleost fish. Progranulin is a secreted growth factor that is involved in normal development and wound-healing processes. We have shown that progranulin promotes the proliferation of retinal precursor cells in mouse retinas. The purpose of this study was to investigate the role played by granulin 1 (grn1) in the retinal regeneration in zebrafish. Methods: We injured the retina of zebrafish with needle puncturing, and the retinas were examined at different times after the injury. We also checked the proliferation and the expression of retinal regeneration-related genes after knockdown of grn1 by electroporation with morpholino oligonucleotides (MO) and intravitreal injection of recombinant grn1. Results: Our results showed that the level of grn1 was highly increased after retinal injury, and it was expressed in various types of retinal cells. A knockdown of grn1 reduced the proliferation of Müller glial cells in zebrafish eyes undergoing retinal regeneration. The knockdown of grn1 also reduced the expression of achaete-scute homolog 1a (ascl1a), an important factor in retinal regeneration. An intravitreal injection of recombinant grn1 led to a proliferation of Müller glial cells and an increase in the expression of retinal regeneration-related genes, such as ascl1a and lin28. Conclusions: These findings suggested that grn1 should be considered as a target for stimulating the dedifferentiation of Müller glial cells and retinal regeneration.


Assuntos
Granulinas/fisiologia , Regeneração/fisiologia , Retina/fisiologia , Degeneração Retiniana/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células , Eletroporação , Inativação Gênica/fisiologia , Granulinas/farmacologia , Imuno-Histoquímica , Morfolinos/toxicidade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/farmacologia , Retina/efeitos dos fármacos , Degeneração Retiniana/etiologia , Degeneração Retiniana/fisiopatologia , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/farmacologia
17.
J Am Heart Assoc ; 7(22): e010690, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30571485

RESUMO

Background The physiological function of ADTRP (androgen-dependent tissue factor pathway inhibitor regulating protein) is unknown. We previously identified ADTRP as coregulating with and supporting the anticoagulant activity of tissue factor pathway inhibitor in endothelial cells in vitro. Here, we studied the role of ADTRP in vivo, specifically related to vascular development, stability, and function. Methods and Results Genetic inhibition of Adtrp produced vascular malformations in the low-pressure vasculature of zebrafish embryos and newborn mice: dilation/tortuosity, perivascular inflammation, extravascular proteolysis, increased permeability, and microhemorrhages, which produced partially penetrant lethality. Vascular leakiness correlated with decreased endothelial cell junction components VE -cadherin and claudin-5. Changes in hemostasis in young adults comprised modest decrease of tissue factor pathway inhibitor antigen and activity and increased tail bleeding time and volume. Cell-based reporter assays revealed that ADTRP negatively regulates canonical Wnt signaling, affecting membrane events downstream of low-density lipoprotein receptor-related protein 6 ( LRP 6) and upstream of glycogen synthase kinase 3 beta. ADTRP deficiency increased aberrant/ectopic Wnt/ß-catenin signaling in vivo in newborn mice and zebrafish embryos, and upregulated matrix metallopeptidase ( MMP )-9 in endothelial cells and mast cells ( MCs ). Vascular lesions in newborn Adtrp -/- pups displayed accumulation of MCs , decreased extracellular matrix content, and deficient perivascular cell coverage. Wnt-pathway inhibition reversed the increased mmp9 in zebrafish embryos, demonstrating that mmp9 expression induced by Adtrp deficiency was downstream of canonical Wnt signaling. Conclusions Our studies demonstrate that ADTRP plays a major role in vascular development and function, most likely through expression in endothelial cells and/or perivascular cells of Wnt-regulated genes that control vascular stability and integrity.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Esterases/fisiologia , Lipoproteínas/fisiologia , Proteínas de Membrana/fisiologia , Neovascularização Fisiológica , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Vasos Sanguíneos/embriologia , Western Blotting , Esterases/genética , Feminino , Imunofluorescência , Técnicas de Inativação de Genes , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
18.
PLoS Genet ; 14(12): e1007821, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30540754

RESUMO

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.


Assuntos
Anemia de Fanconi/genética , Peixe-Zebra/genética , Animais , Sistemas CRISPR-Cas , Dano ao DNA , Anemia de Fanconi/fisiopatologia , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Mutação da Fase de Leitura , Técnicas de Inativação de Genes , Humanos , Masculino , Fenótipo , Processamento de RNA/genética , Processos de Determinação Sexual/genética , Processos de Determinação Sexual/fisiologia , Desenvolvimento Sexual/genética , Desenvolvimento Sexual/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
19.
Science ; 362(6417)2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30467143

RESUMO

The vertebrate body is formed by cell movements and shape change during embryogenesis. It remains undetermined which maternal signals govern the formation of the dorsal organizer and the body axis. We found that maternal depletion of huluwa, a previously unnamed gene, causes loss of the dorsal organizer, the head, and the body axis in zebrafish and Xenopus embryos. Huluwa protein is found on the plasma membrane of blastomeres in the future dorsal region in early zebrafish blastulas. Huluwa has strong dorsalizing and secondary axis-inducing activities, which require ß-catenin but can function independent of Wnt ligand/receptor signaling. Mechanistically, Huluwa binds to and promotes the tankyrase-mediated degradation of Axin. Therefore, maternal Huluwa is an essential determinant of the dorsal organizer and body axis in vertebrate embryos.


Assuntos
Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Herança Materna/genética , Proteínas de Membrana/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , beta Catenina/metabolismo , Animais , Proteína Axina/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteólise , Via de Sinalização Wnt , Proteínas de Xenopus/genética , Xenopus laevis/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
20.
Dev Cell ; 47(3): 319-330.e5, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30399334

RESUMO

Myelin allows for fast and efficient axonal conduction, but much remains to be determined about the mechanisms that regulate myelin formation. To investigate the genetic basis of myelination, we carried out a genetic screen using zebrafish. Here, we show that the lysosomal G protein RagA is essential for CNS myelination. In rraga-/- mutant oligodendrocytes, target genes of the lysosomal transcription factor Tfeb are upregulated, consistent with previous evidence that RagA represses Tfeb activity. Loss of Tfeb function is sufficient to restore myelination in RagA mutants, indicating that hyperactive Tfeb represses myelination. Conversely, tfeb-/- single mutants exhibit ectopic myelin, further indicating that Tfeb represses myelination during development. In a mouse model of de- and remyelination, TFEB expression is increased in oligodendrocytes, but the protein is localized to the cytoplasm, and hence inactive, especially during remyelination. These results define essential regulators of myelination and may advance approaches to therapeutic remyelination.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Bainha de Mielina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Endossomos/metabolismo , Proteínas de Homeodomínio/genética , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/fisiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
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